1. Field of the Invention
This invention relates to a method of the quantitative determination of antigens or antibodies. More specifically, it relates to a method for quantitative analysis for an antigen (or an antibody) which comprises allowing said antigen (or antibody) to react with an antibody (or antigen) supported on an insoluble carrier of fine particle size, irradiating light onto the resultant antigen-antibody complex, and measuring its absorbance at a specific wavelength; particularly a method that can measure the amount of an antigen or antibody in samples taken from living bodies simply and at a high sensitivity.
2. Description of the Prior Art
In the medical field, measuring the concentration of antigens or antibodies in samples taken from living bodies has recently been an important item for the diagnosis of diseases. Particularly, there has been a great demand for the development of a high-sensitivity method for quantitative analysis of those components which are present in samples (for example, the blood) in minute amounts, such as CRP (C-reactive protein) which is an acute reactive substance and AFP (.alpha.-fetoprotein) which is a tumor marker.
The method conventionally used for the quantitative analysis of antigens and antibodies comprises dispersing latex particles of carrier supporting an antibody (or antigen) in a solvent, allowing an antigen (or antibody) to react with said particles, and measuring the increase in turbidity (or absorbance) of the dispersion caused by the antigen-antibody reaction at a wavelength in the range from 600 to 2400 nm, thereby determining the amount of said antigen (or antibody) (Japanese Examined Patent Publication No. 11575/1983).
Another method has been developed recently, which comprises supplying a dispersion containing agglutinated latex particles obtained from an antigen-antibody reaction to a sheath flow so as to make a flow of individual pieces of agglomerates, and analyzing the degree of agglutination by the light scattering method using a laser beam as light source, thereby determining the antigen (or antibody).
However, the above-mentioned methods have the following disadvantages.
In the former method, the change in absorbance due to latex agglutination is very small compared with the absorbance of the latex dispersion itself. If it is attempted to increase the change in absorbance by properly selecting the wavelength for measurement, the absorbance of the latex dispersion itself also tends to increase. Hence, the end-point assay (subtracting the absorbance of latex dispersion itself from the absorbance measured a sufficient period of time after the start of reaction) is difficult to adopt, and the two-point assay (measuring the change in absorbance at two-points over prescribed periods of time after the start of the antigen-antibody reaction) or rate assay (measuring the velocity of the change in absorbance), has to be employed. As a result, an automated analyzer must be used which automatically controls the operations from sample and reagent pipetting to absorbance measurement. In addition, the above-mentioned two-point assay requires a large quantity of costly latex reagent because a lower latex concentration results in lower agglutination speed and lower sensitivity.
Another problem associated with this method is that the change in absorbance is not determined by the degree of latex agglutination alone, because absorbance depends on both the number and size of the particles contained. As shown in FIG. 13, cases are known in which, although latex agglutination proceeds with increasing antigen concentration, absorbance begins to decline when the concentration reaches a certain level. Large errors are unavoidable in these cases.
These disadvantages have been eliminated in the latter method (the light scattering method); the result of measurement is dependent only upon the degree of latex agglutination, the end-point assay can be adopted, latex agglutination proceeds and sensitivty becomes higher with increasing reaction time, and sensitivty remains high even when the latex concentration is decreased. However, the problem is that an exclusive apparatus has to be used, because the flow channel must be of a sheath flow structure and a laser beam must be used as the light source to detect scattered light from individual particles.
This invention provides a simple method for measuring the concentration of an antigen (or an antibody) by a kind of end-point assay using a versatile spectrophotometer without having to employ any exclusive apparatus.